Bovine spongiform encephalopathy and risk to health.
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Animals↗
Biomedical subjects
J E Bell
Publications and source records attributed to J E Bell.
From slow virus to prion.
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Creutzfeldt-Jakob Syndrome↗
Western blotting analysis in patients with MS using human brain vessels as antigen.
Serum samples from patients suffering from multiple sclerosis, other neurological diseases and normal controls were screened by "western blotting" for antibody directed against proteins of human brain vessels purified from a post mortem brain. A small number of sera contained autoantibodies against some of the proteins of the brain vessels, particularly in patients suffering from MS, epilepsy and migraine. The significance of these results is discussed.
Antibody Specificity↗
The partial amino acid sequence of the NAD(+)-dependent glutamate dehydrogenase of Clostridium symbiosum: implications for the evolution and structural basis of coenzyme specificity.
The amino acid sequence is reported for CNBr and tryptic peptide fragments of the NAD(+)-dependent glutamate dehydrogenase of Clostridium symbiosum. Together with the N-terminal sequence, these make up about 75% of the total sequence. The sequence shows extensive similarity with that of the NADP(+)-dependent glutamate dehydrogenase of Escherichia coli (52% identical residues out of the 332 compared) allowing confident placing of the peptide fragments within the overall sequence. This demonstrated sequence similarity with the E. coli enzyme, despite different coenzyme specificity, is much greater than the similarity (31% identities) between the GDH's of C. symbiosum and Peptostreptococcus asaccharolyticus, both NAD(+)-linked. The evolutionary implications are discussed. In the 'fingerprint' region of the nucleotide binding fold the sequence Gly X Gly X X Ala is found, rather than Gly X Gly X X Gly. The sequence found here has previously been associated with NADP+ specificity and its finding in a strictly NAD(+)-dependent enzyme requires closer examination of the function of this structural motif.
Amino Acid Sequence↗
Developmental profile of plasma proteins in human fetal cerebrospinal fluid and blood.
Total protein, alphafetoprotein, albumin, prealbumin, alpha-1-antitrypsin, transferrin and ceruloplasmin levels were measured in samples of human fetal and neonatal cerebrospinal fluid (CSF) (97 cases), obtained between 12 and 41 weeks of gestation. In 39 of these cases (13 to 40 weeks gestation) plasma was also available for comparative analysis. CSF was collected from lateral ventricles in the first half of gestation and from the lumbar region in the second. Since these CSF samples were obtained from different sites, the protein levels in the lateral ventricle (early) samples could not be compared directly with those in the lumbar (later) samples. However, the mean protein levels in the lumbar samples were lower than those in the ventricular samples, which is in accord with the decline in CSF protein levels described in maturing animal fetuses. Despite a wide scatter of results, particularly in the first half of gestation, significant decline in the level of CSF alphafetoprotein was demonstrated during both first and second halves of gestation, and of albumin and prealbumin in the second half. No sex differences were found except for ceruloplasmin in lumbar CSF later in gestation, when males had higher levels than females. In fetal plasma, protein levels increased with increasing gestation apart from alphafetoprotein and prealbumin which both declined progressively. CSF/plasma ratios were dissimilar for different proteins, and changed with increasing gestation. These findings support the concept that the human fetal blood brain barrier matures early.
Blood Proteins↗
Dehydrogenase binding by tiazofurin anabolites.
Thiazole-4-carboxamide adenine dinucleotide (TAD) is the active anabolite of the new antitumor agent tiazofurin (NSC 286193). TAD is an analogue of NAD in which the nicotinamide ring has been replaced by a thiazole-4-carboxamide heterocycle. TAD putatively acts by inhibition of inosine monophosphate dehydrogenase (IMPd). In this study it is shown that TAD is a competitive inhibitor, with respect to NAD, of mammalian glutamate, alcohol, lactate, and malate dehydrogenases. TAD binds to these enzymes with 1-2 orders of magnitude less affinity than it binds to IMPd. Computer modeling studies suggest that dehydrogenase binding by TAD occurs at the regular cofactor site, the thiazole-4-carboxamide group mimicking the steric and hydrogen-bonding properties of the nicotinamide ring. Noncompetitive kinetics of TAD inhibition of the target enzyme IMPd are potentially due to a reverse order of addition of substrate and cofactor from that observed in the dehydrogenases studied here. The weaker binding of TAD to these dehydrogenases may be due to their inability to preserve a close sulfur-oxygen contact in the bound inhibitor.
Adenine Nucleotides↗
Glial and neuronal differentiation in the human fetal brain 9-23 weeks of gestation.
Nineteen human fetal brains ranging from 9-23 weeks of gestation were examined immunocytochemically for evidence of glial and neuronal differentiation. Radial glia were positive for vimentin and glial fibrillary acidic protein (GFAP) throughout the age range. S100-positive cells which were presumed to be astrocytes were present from 9 weeks; they were always more widespread in the cerebrum and the brainstem than GFAP-positive mature astrocytes, which could be detected with certainty only at 14 weeks. Carbonic anhydrase II (CA II)-positive oligodendrocytes were present in the brainstem in small numbers from 17 weeks. Neuronal fibre tracts in the cerebrum were positive for 160 kD phosphorylated neurofilament protein (BF10) from 9 weeks, but negative for 200 kD phosphorylated neurofilament protein (RT97) and for 70 and 200 kD non-phosphorylated neurofilament protein (NFP) whereas most tracts in the brainstem were positive for BF10 from 9 weeks and positive for the other neurofilament proteins from 14 weeks. Corticospinal tracts differed in remaining negative for neurofilament proteins other than BF10, which showed positive reaction throughout. Perikarya of differentiated neurons in all areas of the brain were neurofilament-negative but neuron specific enolase (NSE)-positive. Germinal eminence cells were focally vimentin-positive from 15 weeks, focally GFAP-positive from 17 weeks, and negative for all NFP and for NSE. The value of a short fixation time and pretreatment with trypsin in the immunocytochemical demonstration of GFAP is stressed.
Brain↗
Glutathione S-transferase in human brain.
The glutathione S-transferases are a complex group of multifunctional enzymes which may detoxify a wide range of toxic substances including drugs and carcinogens. Different isoenzymes vary in substrate specificity, tissue distribution and level of expression during development. Following reports of cell-specific and age-dependent expression in rat brain we have studied, immunohistochemically, expression of the Pi and Alpha class isoenzymes in 10 adult and 21 human fetal brains. Whilst Alpha isoenzyme is expressed only in adult brain, and then only focally, Pi isoenzyme is strongly expressed from as early as 12 weeks gestation. In the adult, expression is localized to choroid plexus, vascular endothelium, ventricular lining cells, pia-arachnoid and astrocytes. In fetal brain, expression is also strong in cells with the morphology of tanycytes and in the cell bodies of radial glia. Neurons are consistently negative. Pi isoenzyme thus localizes to the sites of the blood-CSF barrier, blood-brain barrier, CSF-brain barrier and pia-arachnoid-brain barrier. It is ideally placed to regulate neuronal exposure to potentially toxic substances derived from blood or cerebrospinal fluid. Expression so early in gestation is of significance and may imply a role in protection of the developing human brain.
Adult↗
Hepatic microsomal glucose-6-phosphatase system and sudden infant death syndrome.
Microassay techniques and monospecific antibodies were used to study the hepatic glucose-6-phosphatase system in liver samples from 55 infants who had died suddenly and unexpectedly, including 38 victims of sudden infant death syndrome (SIDS). Raised hepatic glycogen was found in 10, all of whom had a diagnosis of SIDS, and in 1 other infant who was already known to have type 1b glycogen storage disease (deficiency of transport protein T1). Of the 10 infants with raised hepatic glycogen who had a diagnosis of SIDS, 8 had glucose-6-phosphatase deficiency (type 1a glycogen storage disease), and 2 had transport protein T2 deficiency (type 1c glycogen storage disease).
Glucose-6-Phosphatase↗
Interaction of terbium and calcium with chicken cystatin.
The emission intensity of the fluorescent lanthanide, terbium, is shown to be enhanced upon binding to chicken cystatin. Fluorescence titrations indicate the presence of a single high affinity binding site per molecule. Binding of the terbium results in a 29% quenching of the fluorescence of the single tryptophan residue in the molecule. Calcium displaces the terbium from cystatin as judged by the decrease of terbium fluorescence in competition titrations. Similar titrations with magnesium or strontium demonstrate that the metal binding site of cystatin exhibits specificity for calcium or terbium. Analysis of the N-terminal sequence of chicken cystatin suggests the presence of a putative consensus sequence for a metal binding site between residues 13 and 24. Calcium causes a 17% decrease in the tryptophan fluorescence of cystatin, indicating that an induced conformational change accompanies metal binding. The increased quenching observed with terbium appears to be the result of resonance energy transfer from tryptophan to terbium. From the critical distance for energy transfer from tryptophan to terbium, it is estimated that the terbium binding site lies approximately 12 A from the single tryptophan residue in the molecule.
Animals↗
Development of the cerebellum with particular reference to cellular differentiation in the external granular layer.
Immunocytochemical evidence of differentiation in developing human cerebellum is presented in this study. Antibodies to neuron specific enolase, neurofilament protein, glial fibrillary acidic protein, vimentin, cytokeratin, epithelial membrane antigen and lymphoid markers, DLC and Leu 7 were used. The external granular layer showed positivity with neuronal markers between 27 weeks gestation and 4 months postnatal, but was negative for all other markers including glial fibrillary acidic protein. Characteristic staining reactions were noted in the other cerebellar layers. Monoclonal antibodies, UJ13A (pan-neuroectodermal marker) and G10 (localising microtubule-associated protein MAP1x) were also used in a limited number of cryostat sections and were positive and negative, respectively, in the external granular layer. The results of this study are discussed in relation to the theory that the external granular layer may be one source of medulloblastomas.
Biomarkers↗
Differentiation in medulloblastomas and other primitive neuroectodermal tumours.
Fifty-four cases of primitive neuroectodermal tumour (PNET) including 47 medulloblastomas, were examined for evidence of neuronal and glial differentiation, using antibodies to neuron-specific enolase (NSE), neurofilament protein (NF), and glial fibrillary acidic protein (GFAP). In 30 of the cases, antibodies to vimentin, alphafetoprotein, cytokeratin, epithelial membrane antigen and lymphoid markers were also used. Most of the 47 medulloblastomas in the group were NSE positive but NF negative; about half were GFAP positive and three of them were positive for both neuronal markers and for GFAP. Vimentin was demonstrated in four cases and was not always co-expressed with GFAP. Medulloblastomas were negative for all the other markers. Supratentorial PNETs were sometimes positive with neuronal markers but were GFAP negative. The cell specificity of these markers and the interpretation of immunocytochemical findings are discussed in relation to differentiation potential in primitive neuroectodermal tumours.
Adolescent↗
Kinetic properties and regulation of biliverdin reductase.
In kinetic studies of the dual nucleotide enzyme biliverdin reductase, product inhibition patterns obtained with bilirubin as the inhibitor, using either the NADPH-linked reaction at pH 8.7 or the NADH-linked reaction at pH 7.0, are consistent with a random order of substrate addition and product release at either pH. With NAD(P) as the product inhibitor, the inhibition patterns suggest the existence of an enzyme-NAD(P)-biliverdin abortive complex. In the absence of added products, substrate inhibition observed at high biliverdin concentrations is also consistent with such a complex. Direct evidence for the existence of enzyme-NADP-biliverdin abortive complexes has been obtained at both pH 7.0 and 8.7 using the fluorescent NADP analog nicotinamide-1-N-6-ethenoadenine dinucleotide phosphate. Etheno-NADP fluorescence is enhanced in enzyme:etheno-NADP complexes but quenched in enzyme:biliverdin:etheno-NADP complexes. The effects of iron hematoporphyrin and cobalt protoporphyrin on the activity of biliverdin reductase have been examined. Iron-hematoporphyrin is a competitive inhibitor with respect to coenzyme at either pH. Comparison of Km values for biliverdin and Ki values for bilirubin or iron-hematoporphyrin indicate that while the binding of biliverdin is relatively pH independent, bilirubin binds more tightly at pH 7.0 than pH 8.7. Conversely, Fe-hematoporphyrin binds considerably tighter at pH 8.7 than pH 7.0. With cobalt-protoporphyrin, however, activation is observed, suggesting the existence of a regulatory site on biliverdin reductase for porphyrin derivatives. Resonance energy transfer measurements, from the fluorescent coenzyme analog etheno-NADP to biliverdin, iron-hematoporphyrin, or cobalt-protoporphyrin indicate that while the biliverdin site is located approximately 15 A from the etheno moiety of the coenzyme, both porphyrin derivatives are located 23.5 A away. This suggest the existence of a separate porphyrin binding site on biliverdin reductase which, depending upon the nature of the porphyrin binding, can produce activation or inhibition of the enzyme.
Algorithms↗
"Bullet"-shaped head in fetuses with spina bifida: a pointer to the spinal lesion.
A "bullet"-shaped head, characterized by flattening in the parietal regions with pointing of the frontal bones, was noted in ultrasound examinations of fetuses with neural tube defects. In a series of 12 fetuses with spinal defects, this finding was present in nine. This abnormal head shape is a useful pointer to the spinal lesion.
Female↗
3,4,5,6-Tetrahydrophthalic anhydride modification of glutamate dehydrogenase: the construction and activity of heterohexamers.
Modification of glutamate dehydrogenase with 3,4,5,6-tetrahydrophthalic anhydride at pH 8.0 results in the progressive loss of enzymatic activity and a concomitant increase in the negative charge of the protein. Although the rate of inactivation at room temperature is too rapid to allow accurate rate constant determination, modification at 4 degrees C shows that the pseudo-first-order rate constant for inactivation appears to show a saturation effect with increasing reagent concentration, with a maximum of approximately 1 min-1. Control experiments showed that tetrahydrophthalic anhydride was hydrolyzed at a much slower rate, with a pseudo-first-order rate constant of 0.041 min-1. Protection studies indicated that inactivation was decreased by the active site ligands, NADP and 2-oxoglutarate. The extents of inactivation, whether assayed with glutamate at pH 7.0 or norvaline at pH 8.0, were the same. Changes in mobility on native gels and isoelectric point were used to follow the incorporated negative charge resulting from modification. Enzyme modified in the presence of protecting ligands (where activity is maintained) showed mobility changes which suggested that a single site of modification was protected. Modified enzyme incorporated 0.78 mol pyridoxal 5-phosphate less than native enzyme, consistent with modification of lysine-126. Enzyme modified under limiting conditions was shown to have a quaternary structure similar to that of the native enzyme, as judged by crosslinking patterns obtained with dimethylpimelimidate. The modified protein is readily resolved from unmodified protein using an NaCl double gradient elution from DEAE-Sephacel. The modification is reversed with regain of activity by incubation of the modified enzyme at low pH. We have made use of the recently demonstrated ability of guanidine hydrochloride to dissociate the hexamer of glutamate dehydrogenase into trimers that can then be reassociated to construct heterohexamers of glutamate dehydrogenase, in which one trimer of the heterohexamer contains native subunits while the other has been inactivated by the 3,4,5,6-tetrahydrophthalic anhydride modification. The heterohexamer is separated from either native or fully modified hexamers by DEAE-Sephacel chromatography. Significantly, the heterohexamer has little detectable catalytic activity, although activity is regained by reversal of the modification of the one modified trimer in the hexamer. This demonstrates that catalytic site cooperation between trimers in the hexamer of glutamate dehydrogenase is an essential component of the enzymatic activity of this enzyme.
Enzyme Activation↗
Use of immunohistochemical methods in the differential diagnosis between primary cerebellar haemangioblastoma and metastatic renal carcinoma.
The potential role of immunohistochemistry in making the distinction between primary cerebellar haemangioblastoma and metastatic renal carcinoma was investigated by examining the reaction pattern of 10 cerebellar haemangioblastomas (seven women, three men, aged 20-40 years) and 10 primary renal carcinomas (six men, four women, aged 49-82 years) to a panel of epithelial, glial, and neural/neuroendocrine antisera. The tumour cell membranes of the renal carcinomas stained strongly with epithelial membrane antigen (EMA); membrane staining was totally absent in the haemangioblastomas. Strong neurone specific enolase (NSE) and S100 staining were also seen in haemangioblastomas but were more variable than EMA staining in renal carcinomas. It is concluded that a panel of antisera is required to distinguish between histologically similar areas in primary haemangioblastomas and metastatic renal carcinomas, and that while complementing conventional histological techniques, new problems of interpretation result which must be taken into account.
Adult↗
Mammalian blastocyst: transport functions in a developing epithelium.
This article reviews the current state of knowledge concerning morphological and physiological mechanisms important to growth and differentiation of the mammalian blastocyst between compaction and implantation. Morphological processes occur in conjunction with major changes in transport systems that control the movement of substances into and out of the embryo. Compaction is a morphological development that is associated with the formation of an outer squamous epithelium, the trophectoderm, which regulates the composition of the medium bathing the presumptive embryo (the inner cell mass). Implantation involves the interaction of two epithelia, the adhesion between the trophectoderm and the maternal endometrium. Before adhesion, the blastocyst lies free in the uterine fluid and exchanges occur between this fluid and the embryo. Apposition of these epithelia is brought about in part by expansion of the blastocyst and removal of the uterine fluid. Blastocyst physiology is an inherently important field because vectorial transport system development and the genes that regulate it can be studied.
Animals↗
A transfer nuclear Overhauser effect study of coenzyme binding to distinct sites in binary and ternary complexes in glutamate dehydrogenase.
The oxidized coenzyme NAD binds to two sites per subunit of bovine liver glutamate dehydrogenase with equal affinity in the absence of dicarboxylic acid coligands. In the presence of glutarate or 2-oxoglutarate, the affinity to one site is unchanged, but the affinity to the other (presumed to be the active site) is considerably increased and now requires two dissociation constants to describe its saturation. A combination of transfer nuclear Overhauser effects (TRNOE) together with an examination of the slopes of TRNOE time dependence indicates that while NAD is bound in a syn conformation at both binding sites, NADP (which binds only to the active site) is bound in a syn-anti mixture. The existence of N6 to N3' and N6 and N2' and N1' to N3' NOE's with NAD suggests that the two coenzyme binding sites are located near enough to allow intermolecular NOE's. In the presence of 2-oxoglutarate where only binding to the active site is effectively observed, the conformation of either coenzyme is syn. Modeling studies using the distance estimates from the TRNOE results suggest that the nicotinamide ribose approximates a 3'-endo conformation. The absence of evidence for intermolecular NOE's under these conditions indicates that while the active and regulatory NAD sites per subunit are in close proximity, the six active sites per hexamer are located greater than 5 A apart.
Animals↗